Unlike eukaryotes, most bacteria do not have a proteasome, but instead, use other protease complexes such as ClpAP and HslUV for their ATP-dependent proteolysis needs. In Mycobacterium tuberculosis (Mtb), however, proteasome is not only present, but its activity is essential for the pathogen to persist in the macrophages o mammalian lung epithelium. Recent research has firmly established that the mycobacterial proteasome is a druggable target and that inhibiting its activity could kill the bacteria. Surprisingly, Mtb uses a ubiquitin (Ub)-like tag, the prokaryotic ubiquitin-like protein (Pup), for delivering the doomed protein for proteasomal degradation. Despite functional similarities between Pup and Ub systems, recent work has revealed fundamental differences between these two conjugation pathways, and between Ub and Pup themselves. Therefore, the prokaryotes and eukaryotes have developed parallel but distinct mechanisms to regulate the protein stability by proteasomes. During the previous funding period, we have successfully addressed several important questions centered on the Mtb 20S proteasome assembly, gate closure and opening mechanism, and how the proteolytic activity is inhibited by the general or Mtb-specific proteasomal inhibitors. We have also revealed that the proteasomal ATPase Mpa recognizes and recruits the pupylated protein substrates via a binding-induced folding mechanism. In the next funding cycle, we will continue to study Mtb proteasome inhibition by novel compounds, and to understand structure and function of the Mtb Pup-proteasome pathway.